Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory.

Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here, we address this knowledge gap using a recent host-range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a possible mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites.

Bridging the Gap Between Single-Strain and Community-Level Plant-Microbe Chemical Interactions.

Although the influence of microbiomes on the health of plant hosts is evident, specific mechanisms shaping the structure and dynamics of microbial communities in the phyllosphere and rhizosphere are only beginning to become clear. Traditionally, plant-microbe interactions have been studied using cultured microbial isolates and plant hosts but the rising use of 'omics tools provides novel snapshots of the total complex community in situ. Here, we discuss the recent advances in tools and techniques used to monitor plant-microbe interactions and the chemical signals that influence these relationships in above- and belowground tissues. Particularly, we highlight advances in integrated microscopy that allow observation of the chemical exchange between individual plant and microbial cells, as well as high-throughput, culture-independent approaches to investigate the total genetic and metabolic contribution of the community. The chemicals discussed have been identified as relevant signals across experimental spectrums. However, mechanistic insight into the specific interactions mediated by many of these chemicals requires further testing. Experimental designs that attempt to bridge the gap in biotic complexity between single strains and whole communities will advance our understanding of the chemical signals governing plant-microbe associations in the rhizosphere and phyllosphere.

Bacterial communities of the Salvia lyrata rhizosphere explained by spatial structure and sampling grain.

Advancements in molecular technology have reduced the constraints that the grain of observation, or the spatial resolution and volume of the sampling unit, has on the characterization of plant-associated microbiomes. With discrete ecological sampling and massive parallel sequencing, we can more precisely portray microbiome community assembly and microbial recruitment to host tissue over space and time. Here, we differentiate rarefied community richness and relative abundance in bacterial microbiomes of Salvia lyrata dependent on three spatial depths, which are discrete physical distances from the soil surface within the rhizosphere microhabitat as a proxy for the root system zones. To assess the impact of sampling grain on rarefied community richness and relative abundance, we evaluated the variation of these metrics between samples pooled prior to DNA extraction and samples pooled after sequencing. A distance-based redundancy analysis with the quantitative Jaccard distance revealed that rhizosphere microbiomes vary in richness between rhizosphere soil depths. At all orders of diversity, rarefied microbial richness was consistently lowest at the deepest samples taken (approximately 4 cm from soil surface) in comparison with other rhizosphere soil depths. We additionally show that finer grain sampling (i.e., three samples of equal volume pooled after sequencing) recovers greater microbial richness when using 16S rRNA gene sequencing to describe microbial communities found within the rhizosphere system. In summary, to further elucidate the extent host-specific microbiomes assemble within the rhizosphere, the grain at which bacterial communities are sampled should reflect and encompass fine-scale heterogeneity of the system.

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly.

BACKGROUND: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. RESULTS: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. CONCLUSION: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly.

Defining the core Arabidopsis thaliana root microbiome.

Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation. Colonization of the root occurs despite a sophisticated plant immune system, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant-microbe interactions derived from complex soil communities.

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria.

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant-microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed-associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed-bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed-bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root-frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

Plant myo-inositol transport influences bacterial colonization phenotypes.

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in seedlings of the model plant Arabidopsis thaliana to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound has been linked to increased host colonization, we identify bacterial phenotypes that occur in both catabolism-dependent and -independent manners, suggesting that myo-inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggest host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite myo-inositol.

A continental-scale survey of Wolbachia infections in blue butterflies reveals evidence of interspecific transfer and invasion dynamics.

Infections by maternally inherited bacterial endosymbionts, especially Wolbachia, are common in insects and other invertebrates but infection dynamics across species ranges are largely under studied. Specifically, we lack a broad understanding of the origin of Wolbachia infections in novel hosts, and the historical and geographical dynamics of infections that are critical for identifying the factors governing their spread. We used Genotype-by-Sequencing data from previous population genomics studies for range-wide surveys of Wolbachia presence and genetic diversity in North American butterflies of the genus Lycaeides. As few as one sequence read identified by assembly to a Wolbachia reference genome provided high accuracy in detecting infections in host butterflies as determined by confirmatory PCR tests, and maximum accuracy was achieved with a threshold of only 5 sequence reads per host individual. Using this threshold, we detected Wolbachia in all but 2 of the 107 sampling localities spanning the continent, with infection frequencies within populations ranging from 0% to 100% of individuals, but with most localities having high infection frequencies (mean = 91% infection rate). Three major lineages of Wolbachia were identified as separate strains that appear to represent 3 separate invasions of Lycaeides butterflies by Wolbachia. Overall, we found extensive evidence for acquisition of Wolbachia through interspecific transfer between host lineages. Strain wLycC was confined to a single butterfly taxon, hybrid lineages derived from it, and closely adjacent populations in other taxa. While the other 2 strains were detected throughout the rest of the continent, strain wLycB almost always co-occurred with wLycA. Our demographic modeling suggests wLycB is a recent invasion. Within strain wLycA, the 2 most frequent haplotypes are confined almost exclusively to separate butterfly taxa with haplotype A1 observed largely in Lycaeides melissa and haplotype A2 observed most often in Lycaeides idas localities, consistent with either cladogenic mode of infection acquisition from a common ancestor or by hybridization and accompanying mutation. More than 1 major Wolbachia strain was observed in 15 localities. These results demonstrate the utility of using resequencing data from hosts to quantify Wolbachia genetic variation and infection frequency and provide evidence of multiple colonizations of novel hosts through hybridization between butterfly lineages and complex dynamics between Wolbachia strains.

mSphere of Influence: Peering through a Keyhole into the Unseen World.

Sarah Lebeis studies the assembly and function of plant microbiomes. In this mSphere of Influence article, she reflects on how the paper "Functional Overlap of the Arabidopsis Leaf and Root Microbiota" (Y. Bai, D. B. Müller, G. Srinivas, R. Garrido-Oter, et al., Nature 528:364-369, 2015, https://doi.org/10.1038/nature16192) provided a roadmap for how large culture collections composed of well-characterized bacterial isolates provide essential resources to test hypotheses concerning microbial communities.

Distinguishing nutrient-dependent plant driven bacterial colonization patterns in alfalfa.

To understand factors that influence the assembly of microbial communities, we inoculated Medicago sativa with a series of nested bacterial synthetic communities and grew plants in distinct nitrogen concentrations. Two isolates in our eight-member synthetic community, Williamsia sp. R60 and Pantoea sp. R4, consistently dominate community structure across nitrogen regimes. While Pantoea sp. R4 consistently colonizes plants to a higher degree compared to the other six organisms across each community and each nutrient level, Williamsia sp. R60 exhibits nutrient specific colonization differences. Williamsia sp. R60 is more abundant in plants grown at higher nitrogen concentrations, but exhibits the opposite trend when no plant is present, indicating plant-driven influence over colonization. Our research discovered unique, repeatable colonization phenotypes for individual microbes during plant microbiome assembly, and identified alterations caused by the host plant, microbes, and available nutrients.

Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88(-/-) mice, IL-1R(-/-) mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R(-/-) mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.

Microbial Ecology: How to Fight the Establishment.

Creating microbial consortia capable of consistently producing desired qualities requires a detailed understanding of community interactions. A new paper demonstrates the role of historical contingency in Arabidopsis thaliana leaf-microbiome formation using an adaptable experimental approach, which could be applied to other host organisms.

Genome-Resolved Proteomic Stable Isotope Probing of Soil Microbial Communities Using 13CO2 and 13C-Methanol.

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from 13CO2 to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived 13C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. 13C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 13C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 13C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Greater than the sum of their parts: characterizing plant microbiomes at the community-level.

Specific subsets of microbes are capable of assembly into plant-associated communities that influence the fitness of both the host and the microbes. While there is a large spectrum of plant phenotypes cause by microbes, the microbial community members benefit from living in protected and nutrient rich plant-associated environments. Recent advances in '-omics' technologies have provided researchers with the ability to identify and assign functions to even unculturable microbes inhabiting both above-ground and below-ground plant tissues. Thus, we are beginning to unravel the molecular mechanisms of microbiome assembly and activities that contribute to overall plant health, not only for individuals, but also at the community-level.

Exercising influence: distinct biotic interactions shape root microbiomes.

Root microbiomes are formed from diverse microbial soil settings with extraordinary consistency, suggesting both defined mechanisms of assembly and specific microbial activity. Recent improvements in sequencing technologies, data analysis techniques, and study design, allow definition of the microbiota within these intimate and important relationships with increasing accuracy. Comparing datasets provides powerful insights into the overlap of plant microbiomes, as well as the impacts of surrounding plants and microbes on root microbiomes and long-term soil conditioning. Here we address how recent studies tease apart the impact of various biotic interactions, including: plant-plant, plant-microbe, and microbe-microbe on root microbiome composition.

PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.

Immune systems distinguish "self" from "nonself" to maintain homeostasis and must differentially gate access to allow colonization by potentially beneficial, nonpathogenic microbes. Plant roots grow within extremely diverse soil microbial communities but assemble a taxonomically limited root-associated microbiome. We grew isogenic Arabidopsis thaliana mutants with altered immune systems in a wild soil and also in recolonization experiments with a synthetic bacterial community. We established that biosynthesis of, and signaling dependent on, the foliar defense phytohormone salicylic acid is required to assemble a normal root microbiome. Salicylic acid modulates colonization of the root by specific bacterial families. Thus, plant immune signaling drives selection from the available microbial communities to sculpt the root microbiome.

The potential for give and take in plant-microbiome relationships.

Mutualistic microbes present in plant-associate microbial communities provide a variety of benefits for their host, including reciprocal exchange of nutrients and/or protection from biotic and abiotic environmental stresses. Plant microbiomes have remarkably robust composition in comparison to the complex and dynamic microbial environments from which they form, suggesting finely tuned discrimination by the plant host. Here the intersection between the plant immune system and microbiomes will be explored, both as a possible means of shaping community membership and as a consequence elicited by certain colonizing microbes. Notably, the advent of massive parallel sequencing technologies allows the investigation of these beneficial microbial functions within whole community settings, so we can now ask how engagement of the immune response influences subsequent microbial interactions. Thus, we are currently poised for future work defining how the plant immune system impacts microbiomes and consequently host health, allowing us to better understand the potential of plant productivity optimization within complex microbial surroundings.

Aligning antimicrobial drug discovery with complex and redundant host-pathogen interactions.

Drug-resistant microorganisms pose an enormous threat to public health. Here we provide examples of experimental approaches that offer novel ways to think about drug development considering the complexity inherent to host-pathogen interactions. Emergent themes include (1) targeting pathogenicity rather than microbial growth, (2) targeting the host or host-pathogen interface rather than the pathogen, (3) facilitating pathogen-specific immune responses, and (4) utilizing systems-based approaches to identify new drug targets and validate drug efficacy. We posit that together these approaches may allow identification of drugs that disrupt pathogenesis and allow the immune system time to protect, but do not easily engender resistance.

Protective and destructive innate immune responses to enteropathogenic Escherichia coli and related A/E pathogens.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli (O157:H7) and Citrobacter rodentium are classified as attaching and effacing (A/E) pathogens based on their ability to adhere to intestinal epithelium, destroy microvilli and induce pedestal formation at the site of infection. A/E bacterial infections also cause acute diarrheal episodes and intestinal inflammation. The use of model systems has led to an understanding of the innate immune response to A/E pathogens. The innate immune system plays a protective role, initiating a productive antibody response, directly killing bacteria and inducing repair mechanisms following tissue damage caused by infection. However, hyperactivation of the innate immune system can have negative consequences, including exacerbated tissue destruction following neutrophil infiltration. Here we review how innate immune cell types, including neutrophils, macrophages and dendritic cells, orchestrate both protective and destructive responses. Such information is crucial for the development of therapeutics that can mitigate destructive inflammatory responses while accentuating those that are protective.